(i) Field of the invention
This invention relates to a diagnostic kit useful in establishing the existence or potential existence of materno-fetal immunoincompatibility in women. The invention also relates to a method of preparing the kit, its constituents and to a method of using the kit.
(ii) Description of the Prior Art
In normal pregnancy, a fetus, despite being an immunologically separate entity from its mother, is not attacked by its mother's immune system. Thus a "host versus graft reaction" (HVGR) as in rejection of transplanted organs, does not occur.
In other words, the antigenic determinants of the fetus are not recognized by the mother's immune system. This is because the (blood) serum of the mother normally contains blocking substances which partially block the fetus' minor histocompatibility antigenic determinants rendering them "invisible" to the mother's immune system.
The maternal serum has this effect on several fetal antigens some of which are the class II histocompatibility antigens (otherwise known as HLA-Ds).
In normal pregnancy, fetal cells show diminished expression of these antigens because they are wholly or partially blocked by said blocking agents.
It has been observed that spontaneous miscarriages and certain kinds of infertility in women as well as the risk of preterm delivery result from the patients' inability to produce such blocking substances, i.e. the absence or reduction in amount of these substances in certain pathological cases causes the said miscarriages, infertility, or preterm delivery.
Patients suffering from past miscarriages or infertility and wishing to become pregnant or patients whose fetus is at risk, are presently treated by injection of mononuclear blood cells taken from their respective mates or from other healthy (male) donors.
Patients showing signs of impending preterm delivery (e.g. contractions) presently undergo intensive conservative treatment (e.g. bedrest and perfusion with beta-mimetic drugs) to avoid premature termination of their pregnancies.
The antigenic incompatibility of the type under discussion is mainly found in couples partly sharing HLA class I or HLA class II antigens.
Clearly, not all infertilities, miscarriages and preterm deliveries are caused by the above-described immunological problem and therefore not all patients can be successfully treated by the above-described injection of blood cells (in the first two cases) or by intensive conservative treatment (in the third case).
It is therefore desirable to have a means of selecting which patients may be susceptible to such treatments.
Such selection is presently done on a clinical basis (i.e. by exclusion) and also after a HLA class I and class II study. In other words, where no obvious organic, homonal or infectious etiology is demonstrated, it is assumed that an immunological insufficiency could be a possible etiology. Thus no positive diagnosis of immunological insufficiency is made, instead only indirect evidence is provided to support such a diagnosis.
Recently Alan BEER at the INSERM/CNRS Materno-Fetal Relationship Colloque held at the Clinique Universitaire Baudelocque on Oct. 18, 1986, in Paris, France, has proposed the use of a mixed lymphocyte reaction (MLR) - see page 38 of IMMUNOLOGY IN OBSTETRICS AND GYNECOLOGY by James R. SCOTT et al, pub. Appleton-Century-Crofts-as a diagnostic tool.
In this test, maternal lymphocytes are mixed with paternal lymphocytes. The HLA-D (class II) antigens on the paternal lymphocytes stimulate proliferation of the maternal lymphocytes. This proliferation is measured by the amount of DNA synthesis occuring in the maternal lymphocyte. In pathological cases DNA synthesis is increased over healthy cases since normally the paternal antigens are blocked by the blocking agent. Thus the test interprets increased DNA synthesis to mean lack, or reduced presence, of the blocking agent i.e. a pathological condition. However, BEER's test proved unreliable as it gave unreproducible results.
Another team, headed by J. F. Mowbray, has proposed a haemoagglutination test. This was described at the 3rd International Congress of Reproductive Immunology in Toronto, Canada, from July 1-5, 1986. However, this test does not measure the inhibitory effect of maternal serum.
In any event, the prior art test procedures all involve living cells in the test materials. This brings many problems such as possible infection of the materials, genetic mutation, inconstant phenotypic characteristics aswell as the concomitant problems of storage and short shelf-life. The above problems often result in unreliable and inconstant results.